The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between redox centers within a protein complex. New approaches to this problem are being explored in a multi-disciplinary project directed by an inorganic chemist and physical biochemist. A new class of ruthenium- labeled proteins have been developed that allow biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. A novel two-step procedure was developed to label cytochrome c at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine. More than ten singly labeled derivatives have now been purified and characterized. One of the most remarkable properties of this ruthenium complex is that it can be photoexcited to a mental-to-ligand charge-transfer state, RuII*, which is a strong reducing agent and can transfer an electron to the heme group FeIII in cytochrome c. The electron transfer rate constants were found to be 14-30 X 106 S-1 for derivatives modified at lysines 13, 27, and 72, which have a separation of 8 to 12 A between the ruthenium and heme groups. These are the largest electron transfer rate constants ever measured for a native heme group in a cytochrome. The rate constants were significantly smaller for derivatives with a larger separation between the ruthenium and heme groups. A new method was also developed to measure the rates of electron transfer within complexes between the ruthenium-cytochrome c derivatives and other proteins. The specific aims for the next granting period will be: 1) to measure the photoinduced electron transfer reactions between the ruthenium and heme groups as a function of temperature and driving force to determine the reorganization barrier of the reaction. 2) to study the dependence of electron transfer on distance and protein medium by comparing the rate constants of derivatives labeled at different lysines on the surface of cytochrome c. 3) to study the intra-complex electron transfer reaction between the ruthenium cytochrome c derivatives and cytochrome oxidase. 4) to study the intra-complex, cytochrome c peroxidase, and cytochrome b5. 5) to develop new chemical modification methods to extend the ruthenium labeling technique to other electron transfer proteins. 6) to study the interaction of Rps. viridis cytochrome c2 with the Rps. viridis photosynthetic reaction center.